Cell surface glycans refer to a vast variety of glycan motifs attached to plasma membrane bound proteins or lipids. These constitute the outermost surface of the cell and are involved in cellular communication and processes like host-pathogen recognition, infection, cellular differentiation, proliferation and migration. Around 700 proteins are required to generate the full diversity of mammalian glycans, which are assembled from ten monosaccharides only: fucose (Fuc), galactose (Gal), glucose (Glc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), glucuronic acid (GlcA), iduronic acid (IdoA), mannose (Man), sialic acid (SA) and xylose (Xyl). The most-widely occurring cancer-associated changes in glycosylation are sialylation, fucosylation, O-glycan truncation, and N- and O-linked glycan branching (Chart 1).
SA are the outermost monosaccharide units on the glycan chains of glycolipids and glycoproteins and often the recognition sites where the pathogens attach. The occurrence of SA has proven to correlate with several disease states such as cardiovascular, and neurological diseases as well as cancer (B. Adamczyk, T. Tharmalingam and P. M. Rudd, Biochimica et Biophysica Acta (BBA)—General Subjects, 2012, 1820, 1347-1353). Clinically, increased sialylation is often associated with invasiveness and poor prognosis of cancer patients. An incomplete synthesis process leads to the biosynthesis of truncated structures, as seen with STn (Chart 1) expression in gastrointestinal and breast cancers. STn (Neu5Acα2-6GalNAcα—O—R) and Tn (GalNAcα—O—R) have attracted wide spread attention as a diagnostic as well as therapeutic target since it is expressed in 80% of human cancers and absent or only weakly expressed in normal tissue. However, it has been difficult to produce IgG antibodies against Tn and ST and IgG antibodies against STn have low affinity and perform poorly in capture strategies. The few lectins available to SA (SNA and MAA) have broad specificities and low affinities, and perform poorly in capture assays.
Conversely, neo-synthesis, commonly observed in advanced stages of cancer, refers to the cancer-associated induction of certain genes involved in the expression of carbohydrate determinants, as seen in the de novo expression of certain antigens, such as sialyl Lewis a (SLea) and sialyl Lewis X, SLex, in many cancers. Polysialic acid is increased sialylation associated with several types of cancers, and frequently expressed in high-grade tumours.
There are two main mammalian SAs, Neu5Ac (N-acetylneuraminic acid) and Neu5Gc (N-glycolylneuraminic acid) (see Chart 1), which differ only by one oxygen atom and is added by the enzyme cytidine monophosphate N-acetylneuraminic acid hydroxylase (CMAH) in the cytosol. Humans lack this enzymatic activity due to an inactivating mutation of the CMAHgene. As this alternative form of SA is not normally present, immunological tolerance fails to develop. As a result, when Neu5Gc incorporation into glycoconjugates occurs after intake from dietary sources, “autoantibodies” to this sugar develop, and these have been proposed to enhance inflammatory pathways associated with cancer initiation. Novel affinity reagents for discriminating these forms as well as linkage specific animal and human glycosylations 2-3 versus 2-6 linked sialic acids are highly demanded.
Analyzing and determining these glycosylation motifs is therefore an important diagnostic goal but the task has proven challenging due to the limited availability of lectins and glycan specific antibodies (N. Fujitani, J.-i. Furukawa, K. Araki, T. Fujioka, Y. Takegawa, J. Piao, T. Nishioka, T. Tamura, T. Nikaido, M. Ito, Y. Nakamura and Y. Shinohara, Proceedings of the National Academy of Sciences, 2013, 110, 2105-2110).
A number of sensitive methods have nevertheless been published that either measure total sialic acid content or different sialic acid types. These methods are costly, laborious and time consuming, and commonly require sophisticated instrumentation as skilled operators for their implementation. They are therefore not well suited for routine applications. Lectin-based assays exist for a few types of sialic acids but assay development is often hampered by their low sensitivity and poor specificity. The detection of sialic acid in sialoglycoprotein or as a free moiety include the use of high performance liquid chromatography (HPLC), gas-chromatography combined with mass spectrometry (GC-MS), nuclear magnetic resonance spectrometry (NMR) and capillary electrophoresis (CE). These methods are complicated by significant requirements for sample preparation, specialised equipment, purification of the target protein and lengthy and complex data analysis for monitoring the sialylation pattern.
This warrants the development of alternative glycan specific receptors which could be used for e.g. cell or tissue imaging, cell sorting, targeted glycomics and cellular glycosylation biomarker analysis or for applications in medicine for instance for targeted drug delivery or the selective inhibition of cell surface interactions. A plethora of low molecular hosts has been systematically designed and conjugated e.g. to fluorescent reporter groups or quantum dots for imaging applications. The most powerful hosts for sialic acid feature two or more orthogonal binding groups, a boronic acid directed towards the diol functionality and a charged or neutral anion receptor directed towards the carboxylate function. Other strong binders are multi-functional incorporating two or more boronic acid groups. The latter engage in a pH dependent reversible esterification with the diols resulting in five or six membered cyclic structures.
It has long been known that monosaccharide selective receptors can be prepared by the technique of molecular imprinting. Wulff et al. reported highly discriminative boronate-based receptors for mannose, fructose and galactose prepared using the monosaccharide templates conjugated to two molecules of vinylbenzeneboronic acid (1). Other researchers later adopted this procedure for the synthesis of sialic acid imprinted bulk polymers or sensor coatings. These boronic acid containing MIPs featured strong template affinity when probed in basic buffer/acetonitrile mixtures (pH 8). A simpler one-pot protocol was subsequently used for the synthesis of glycoprotein selective MIPs (X. Bi and Z. Liu, Analytical Chemistry, 2013, 86, 959-966). Here the boronate monomer is conjugated in situ under base catalysis to the SA containing glycoprotein and subsequently copolymerized with a crosslinking monomer to form the imprinted polymer. The SA imprinted receptors reported so far display only weak binding for the saccharide targets (K<103 M−1) when probed in water and especially under physiological conditions. This contrasts with the most powerful designed hosts (vide supra) where binding is sufficiently strong (>105 M−1) to allow cell based imaging or even cell sorting applications. The latter receptors however are complicated to design for targeting more complex glycans such as disaccharide and higher saccharides. This commonly requires extensive synthetic efforts and testing of a large number of analogs in order to identify strong binders.
Apart from cell or tissue imaging applications such receptors could be used to replace lectins or antibodies in targeted glycomics, in glycan sensors, for enrichments of specific glycan motifs, for cell sorting or in medicine for instance for targeted drug delivery or the selective inhibition of cell surface interactions.
One application concerns the identification and molecular characterization of circulating tumor cells (CTCs) in cancer patients. Characterising these cells is important for understanding the metastatic process and potential therapeutic strategies thereby guiding prognosis and treatment. Progress in this field has been slow which partly is due to the low, “needle in a haystack”, abundance of CTCs. Although CTCs can be reliably detected in patients with metastatic disease, challenges remain to detect early stage, treatable cancers with inferior CTC numbers. The currently dominating techniques to detect CTCs are cytometric assays where cells remain intact preceeded by an initial enrichment step to optimize the probability of rare cell detection. The dominating enrichment technique is based on immunomagnetic separation typically dependent on the epithelial protein marker (EpCam). However, cell surface glycans or circulating O-glycoproteins shed from cancer cells also represent important serum biomarkers for diagnostic and prognostic purposes. Selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays.
Targeted delivery of drugs to tumors represents a significant advance in cancer diagnosis and therapy. Therefore, development of novel tumor-specific ligands or pharmaceutical nanocarriers is highly desirable. These nanocarriers would be loaded with drugs and targeted to specific parts of the body where there is solely diseased tissue, thereby avoiding interaction with healthy tissue. Targeted delivery to specific cell organelles through endocytosis of the delivery vehicle (nanocarrier) offers a more specific targeting especially suited for macromolecular drugs. Selective targeting of such nanocarriers to cells expressing cancer associated glycans is an important goal.